Capillaries are useful in separatory processes of various kinds. Examples of such processes are various forms of electrophoresis, including free-zone or open-tube electrophoresis, isotachophoresis and isoelectric focusing, both with and without electroendosmotic flow. Due to their small internal volume, capillaries can effectively separate extremely small samples, and can do so at high speed. Capillaries are of particular interest in electrophoretic separations since the narrow bore of a capillary promotes rapid heat dissipation outward, which permits the use of high voltages. This provides high speed and efficiency, rendering capillaries particularly useful for analyzing samples of biological interest, particularly mixtures of small peptides, proteins and nucleic acids.
One of the problems in capillary electrophoresis is the flushing of the capillary with buffer or wash solution in between separations. This is generally achieved by pressure differential techniques, which require external apparatus such as a pump or a vacuum line. Such apparatus presents problems in designing systems for the automated analysis of a series of samples, and is generally awkward in terms of its integration with the other components associated with the system. Problems also arise from the limited extent to which one can control the pressure differential and its duration.
A further problem is the loading of the sample, i.e., its placement inside the end of the capillary in preparation for the separation. At present, this is commonly achieved by electrophoretic, electroendosmotic or pressure differential techniques.
In electrophoretic loading, a high voltage is used over a short period of time to transfer the sample from a sample reservoir into the capillary itself. The goal is to move small amounts of all species in the sample a short distance into the capillary. Once this is done, the sample reservoir is replaced with an appropriate buffer solution to permit one to proceed with electrophoretic separation of the loaded species.
In electroendosmotic loading, the fluid is moved primarily by bulk flow into the capillary as the result of an electroendosmotic effect in the capillary. What actually occurs in electroendosmotic loading is a combination of electrophoresis and electroendosmosis, with electroendosmosis having the predominating effect. The disadvantage of electroendosmosis is that it varies from one experiment to the next, causing difficulties in the reproducibility of the sample volume. One can eliminate electroendosmosis as a driving force by applying an appropriate coating to the inside of the capillary. This will leave electrophoresis as the sole driving force.
Electrophoretic loading, however, has its own disadvantages. These arise from the differentials which necessarily exist among the various species in the sample in terms of their response to the electric potential. These differentials affect the distance which the species travel into the capillary during sample loading and thus the amounts of each species entering the capillary. Slowly migrating substances will thus migrate a shorter distance into the tube than will the faster migrating substances. Depending on the loading conditions, therefore, the composition of the applied sample may differ from that of the original sample.
Electrophoretic and electroendosmotic loading both have the further drawback that the current will cause an increase in temperature in the capillary. This fact gives rise to an expansion of the liquid in the capillary, which may cause part of the sample to be displaced backwards out of the capillary in an irreproducible way. This in turn may mean that the amount of sample introduced will vary from one experiment to the next.
Other variables in electrophoresis such as fluctuations in current strength may also enter into consideration. The significance and importance of these variables will vary from one system to the next.
In hydraulic loading, sample introduction is achieved in the same manner as the column flushing referred to above, i.e., either by applying a partial vacuum to the outlet end of the capillary or a positive pressure to the inlet end. The limited extent to which one can control the pressure differential and its duration present even more of a problem here, since they affect the volume of the sample introduced.